Leptospirosis is a neglected global human health problem caused by transmission from reservoir hosts that harbor pathogenic Leptospira species in their kidneys and shed them into the environment via their urine. Our goal is to elucidate the role(s) of the surface-exposed leptospiral immunoglobulin- like (Lig) proteins in mechanisms of leptospiral pathogenesis and immunity. The LigA and LigB proteins exhibit high affinity binding to host ligands and inhibit complement activation, trigger plasminogen, and inhibit fibrin formation. However, initial studies found that ligA and ligB single gene knockout mutants were competent for infection. A recent study by one of our collaborators showed that knocking down expression of both ligA and ligB by targeting their identical promoters with the TALE system resulted in the loss of virulence, indicating that the functions of LigA and LigB are redundant. Despite the large and growing literature on their structure and function, the roles of the Lig proteins in virulence remain poorly understood. The in vitro activities of the Lig proteins suggest that their role is to resist host defense mechanisms. Our overall hypothesis is that discrete segments of the Lig proteins are required at an early step of infection to evade killing by the host. For this project, we will focus on LigB since ligA is missing from a majority of pathogenic Leptospira species. We will evaluate the functions of LigB in the context of the bacterial cell and determine which of its domains are responsible for these functions. We will also develop a LigB vaccine that provides cross-protective immunity. To accomplish these goals, we will employ powerful and innovative approaches to track infection including whole animal optical imaging and high-throughput parallel sequencing. These studies are vital for development of effective approaches for protection from and treatment of leptospirosis. Specific Aim 1. When are the Lig proteins critical for infection? The wild-type and TALE-lig knockdown strains of L. interrogans will be engineered to express bioluminescence. Hamsters will be infected with the bioluminescent strains, and infection will be monitored with whole animal in vivo imaging to determine when and where infection is halted when the Lig proteins are not generated. Specific Aim 2. What functions are mediated by LigB expressed on the bacterial cell surface? We will determine the bacterial cell surface functions mediated by the LigB by employing a ?gain-of-function? strategy. We will transform L. biflexa with a ligB plasmid and test the ability of the ?knock-in? strain to adhere to host plasma proteins, interfere with serum killing, activate plasminogen, and slow fibrin clot formation. We will also perform these experiments with the L. interrogans TALE-lig knockdown strain and with the knockdown strain harboring ligB plasmid to determine the contribution of LigB to these pathogenic functions. Specific Aim 3. What regions of LigB are responsible for their virulence properties? We will determine by mutation analysis which segments of LigB contain sites necessary for virulence. LigB variants generated by domain-swapping and alanine-scanning mutagenesis will be expressed from barcoded plasmids in the TALE-lig knockdown strain of L. interrogans. Leptospires expressing the LigB variants will be pooled and inoculated into hamsters. The fitness of the variants will be assessed by high throughput parallel sequencing. LigB variants that reduce fitness in vivo will undergo functional evaluation in vitro. Specifc Aim #4. Does LigB generate cross-protective immunity? Soluble recombinant proteins comprising different segments of LigB will be generated and tested for their immunoprotective potential in the hamster model of acute lethal leptospirosis. Immunized hamsters will be challenged with L. interrogans and L. kirschneri to assess cross-protection. Kidneys will be evaluated by culture, qPCR, serology, and immunohistochemistry to assess renal colonization.